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1.
Circ Heart Fail ; 11(5): e004491, 2018 05.
Article in English | MEDLINE | ID: mdl-29724722

ABSTRACT

BACKGROUND: Coronary artery fluid dynamics may be altered because of the nonphysiological flow seen in continuous-flow left ventricular assist devices (CF-LVADs). Our aim was to study the structure and composition of coronary vessels after CF-LVAD. METHODS AND RESULTS: Coronary arteries were collected from patients with heart failure (HF) at the time of transplantation, of whom 15 were supported with a CF-LVAD before transplant (HF+LVAD group) and 9 were not (HF non-LVAD group). In addition, coronary samples were obtained from 5 nonfailing age-matched donors (nonfailing group). Histological analysis was performed to quantify coronary morphology, composition, vascular fibrosis, and vasa vasorum density. The age and sex mix of the 3 groups were similar, and the mean duration of LVAD support was 213 days. Compared with patients with HF and nonfailing donors, the arteries from patients with HF+LVAD had expansion of the adventitia, breakdown of the internal elastic lamina, and increased adventitial collagen deposition and density of vasa vasorum. CONCLUSIONS: Among patients supported with CF-LVADs, the coronary arteries develop marked remodeling with increased adventitial fibrosis. The physiological consequences of these structural changes are unknown, but it is possible that arterial contractility may be impaired, thus limiting coronary flow reserve and promoting myocardial ischemia. This may contribute to CF-LVAD complications, such as ventricular arrhythmias and right ventricular failure. As more patients receive CF-LVADs and new pump technology attempts to modulate flow profiles and pulsatility, further research is needed to understand the mechanisms and long-term sequela of these changes in coronary arteries and other vascular beds.


Subject(s)
Coronary Vessels/physiopathology , Fibrosis/physiopathology , Heart Failure/physiopathology , Heart-Assist Devices , Adult , Aged , Female , Heart/physiopathology , Humans , Male , Middle Aged , Myocardial Ischemia/physiopathology , Ventricular Function, Left/physiology
2.
JCI Insight ; 3(4)2018 02 22.
Article in English | MEDLINE | ID: mdl-29467331

ABSTRACT

Phosphatase and tensin homolog (PTEN) is an essential regulator of the differentiated vascular smooth muscle cell (SMC) phenotype. Our goal was to establish that PTEN loss promotes SMC dedifferentiation and pathological vascular remodeling in human atherosclerotic coronary arteries and nonatherosclerotic coronary arteries exposed to continuous-flow left ventricular assist devices (CF-LVADs). Arteries were categorized as nonatherosclerotic hyperplasia (NAH), atherosclerotic hyperplasia (AH), or complex plaque (CP). NAH coronary arteries from CF-LVAD patients were compared to NAH coronaries from non-LVAD patients. Intimal PTEN and SMC contractile protein expression was reduced compared with the media in arteries with NAH, AH, or CP. Compared with NAH, PTEN and SMC contractile protein expression was reduced in the media and intima of arteries with AH and CP. NAH arteries from CF-LVAD patients showed marked vascular remodeling and reduced PTEN and α-smooth muscle actin (αSMA) in medial SMCs compared with arteries from non-LVAD patients; this correlated with increased medial collagen deposition. Mechanistically, compared with ApoE-/- mice, SMC-specific PTEN-null/ApoE-/- double-knockout mice exhibited accelerated atherosclerosis progression and increased vascular fibrosis. By microarray and validated quantitative RT-PCR analysis, SMC PTEN deficiency promotes a global upregulation of proinflammatory and profibrotic genes. We propose that PTEN is an antiinflammatory, antifibrotic target that functions to maintain SMC differentiation. SMC loss of PTEN results in pathological vascular remodeling of human arteries.


Subject(s)
Atherosclerosis/pathology , Coronary Vessels/pathology , Heart-Assist Devices/adverse effects , Myocytes, Smooth Muscle/pathology , PTEN Phosphohydrolase/deficiency , Vascular Remodeling , Actins/metabolism , Adult , Aged , Animals , Atherosclerosis/genetics , Cell Differentiation , Coronary Vessels/cytology , Disease Models, Animal , Endothelium, Vascular , Female , Fibrosis , Heart Failure/surgery , Humans , Hyperplasia/pathology , Male , Mice , Mice, Knockout, ApoE , Middle Aged , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/pathology , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism
3.
Nat Commun ; 7: 10830, 2016 Mar 04.
Article in English | MEDLINE | ID: mdl-26940659

ABSTRACT

Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN-SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.


Subject(s)
Myocytes, Smooth Muscle/physiology , PTEN Phosphohydrolase/metabolism , Serum Response Factor/metabolism , Animals , Cell Differentiation , Cells, Cultured , Gene Expression Regulation/physiology , Humans , Male , Mice , Mice, Knockout , Mice, Transgenic , Muscle, Smooth, Vascular/cytology , PTEN Phosphohydrolase/genetics , Rats , Rats, Sprague-Dawley , Serum Response Factor/genetics
4.
Cell Host Microbe ; 14(3): 329-39, 2013 Sep 11.
Article in English | MEDLINE | ID: mdl-24034618

ABSTRACT

Understanding gut microbiota alterations associated with HIV infection and factors that drive these alterations may help explain gut-linked diseases prevalent with HIV. 16S rRNA sequencing of feces from HIV-infected individuals revealed that HIV infection is associated with highly characteristic gut community changes, and antiretroviral therapy does not consistently restore the microbiota to an HIV-negative state. Despite the chronic gut inflammation characteristic of HIV infection, the associated microbiota showed limited similarity with other inflammatory states and instead showed increased, rather than decreased, diversity. Meta-analysis revealed that the microbiota of HIV-infected individuals in the U.S. was most similar to a Prevotella-rich community composition typically observed in healthy individuals in agrarian cultures of Malawi and Venezuela and related to that of U.S. individuals with carbohydrate-rich, protein- and fat-poor diets. By evaluating innate and adaptive immune responses to lysates from bacteria that differ with HIV, we explore the functional drivers of these compositional differences.


Subject(s)
Bacteria/classification , Biota , Dysbiosis , Gastrointestinal Tract/microbiology , HIV Infections/complications , Bacteria/growth & development , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Feces/microbiology , Humans , Molecular Sequence Data , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA
5.
Diabetes Technol Ther ; 11(4): 227-33, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19344197

ABSTRACT

BACKGROUND: Most insulin autoantibody assays for both human and animal models are in a radioassay format utilizing (125)I-insulin, but despite the radioassay format international workshops have documented difficulty in standardization between laboratories. There is thus a need for simpler assay formats that do not utilize radioactivity, yet retain the high specificity and sensitivity of radioassays. METHODS: To establish an easier enzyme-linked immunosorbent assay (ELISA) for insulin autoantibodies of non-obese diabetic (NOD) mice, we used an ELISA format, competition with unlabeled insulin, europium-avidin, and time-resolved fluorescence detection (competitive europium insulin autoantibody assay). RESULTS: The competitive europium assay of insulin autoantibodies when applied to sera from NOD mice had high sensitivity and specificity (92% sensitivity, 100% specificity) compared to our standard insulin autoantibody radioassay (72% sensitivity, 100% specificity) in analyzing blind workshop sera. It is noteworthy that though the assay has extremely high sensitivity for murine insulin autoantibodies and utilizes human insulin as target autoantigen, human sera with high levels of insulin autoantibodies are not detected. CONCLUSIONS: Our results clearly indicate that low levels of insulin autoantibodies can be detected in an ELISA-like format. Combining a europium-based ELISA with competition with fluid-phase autoantigen can be applicable to many autoantigens to achieve high specificity and sensitivity in an ELISA format.


Subject(s)
Autoantibodies/blood , Diabetes Mellitus, Type 1/immunology , Insulin Antibodies/blood , Animals , Autoantigens/immunology , Biotinylation , Diabetes Mellitus, Type 1/blood , Enzyme-Linked Immunosorbent Assay/methods , Europium , Humans , Insulin/deficiency , Insulin/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Sensitivity and Specificity , Streptavidin
6.
Am J Gastroenterol ; 104(1): 154-63, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19098864

ABSTRACT

OBJECTIVES: Measurement of transglutaminase autoantibodies (TGAA) is considered to be the most efficient single serologic test for celiac disease (CD) by the American Gastroenterological Association Institute. We hypothesized that a large international collaborative effort toward improving and standardizing TGAA measurement is both feasible and necessary. The primary aim of this workshop is to compare TGAA assays among various research and clinical laboratories to examine assay concordance and improve (and eventually standardize) the TGAA assay. METHODS: A total of 20 laboratories (5 commercial laboratories, 15 research and clinical laboratories) participated that included enzyme-linked immunosorbent assay (ELISA) and radiobinding assays. A total of 150 serum samples were distributed to each laboratory, with each laboratory receiving an equal aliquot that was coded and blinded, composed of 100 healthy control sera and 50 CD sera. RESULTS: Laboratory sensitivity ranged from 69% to 93% and specificity ranged from 96% to 100%. By receiver operator characteristic analysis, the area under the curve (C index) ranged from 0.9488 to 0.9904. When analyzing for linear correlation, r-squared was as high as 0.8882 but as low as 0.4244 for the celiac samples between different laboratories performing ELISA. CONCLUSIONS: This transglutaminase autoantibody workshop allows for larger-scale international participation for the purposes of improving and eventually standardizing the TGAA assay with subsequent workshops.


Subject(s)
Autoantibodies/blood , Celiac Disease/diagnosis , Laboratories/standards , Transglutaminases/immunology , Biomarkers/blood , Enzyme-Linked Immunosorbent Assay/standards , Humans , International Cooperation , ROC Curve , Radioligand Assay/standards , Sensitivity and Specificity
7.
Proc Natl Acad Sci U S A ; 105(29): 10090-4, 2008 Jul 22.
Article in English | MEDLINE | ID: mdl-18626021

ABSTRACT

A fundamental question is what are the molecular determinants that lead to spontaneous preferential targeting of specific autoantigens in autoimmune diseases, such as the insulin B:9-23 peptide sequence in type 1 diabetes. Anti-insulin B:9-23 T cell clones isolated from prediabetic NOD islets have a conserved Valpha-segment/Jalpha-segment, but no conservation of the alpha-chain N region and no conservation of the Vbeta-chain. Here, we show that the conserved T cell receptor alpha-chain generates insulin autoantibodies when transgenically or retrogenically introduced into mice without its corresponding Vbeta. We suggest that a major part of the mystery as to why islet autoimmunity develops relates to recognition of a primary insulin peptide by a conserved alpha chain T cell receptor.


Subject(s)
Autoantibodies/biosynthesis , Genes, T-Cell Receptor alpha , Insulin Antibodies/biosynthesis , Insulin/immunology , Adoptive Transfer , Animals , Base Sequence , Clone Cells/immunology , Conserved Sequence , DNA Primers/genetics , Female , Genes, T-Cell Receptor beta , Interferon-gamma/biosynthesis , Islets of Langerhans/immunology , Islets of Langerhans/pathology , Male , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Lymphocytes/immunology
8.
J Pediatr Gastroenterol Nutr ; 45(3): 293-300, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17873740

ABSTRACT

INTRODUCTION: Gliadin proteins play a key role in the pathogenesis of celiac disease; however, as a screen for celiac disease, anti-gliadin antibody testing has been replaced by the more sensitive and specific serological assays for transglutaminase autoantibodies (TGAA). A new generation of anti-gliadin antibody assays has been developed to detect synthetic, deamidated homologous gliadin peptides (DGP) with high sensitivity and specificity. METHODS: Sera were collected prospectively from children with an increased risk for celiac disease as part of an ongoing study at Denver, and studied for the development of celiac autoimmunity. We investigated the high-performance DGP antibody assay in 50 TGAA-positive children both before the development of celiac autoimmunity and following the institution of a gluten-free diet to determine the relationship of DGP antibodies to TGAA. TGAA were measured by an in-house radioassay. RESULTS: DGP antibodies and TGAA parallel each other over the period of years children were studied. DGP antibodies resolved sooner than TGAA in subjects on a gluten-free diet. DGP antibodies appeared earlier than TGAA in 9 children. CONCLUSIONS: Measuring DGP antibodies may be more useful than TGAA in monitoring children on a gluten-free diet. DGP antibodies can precede the appearance of TGAA in some at-risk children.


Subject(s)
Autoantibodies , Celiac Disease/diagnosis , Gliadin/administration & dosage , Gliadin/immunology , Transglutaminases/immunology , Adolescent , Autoantibodies/analysis , Celiac Disease/blood , Celiac Disease/genetics , Child , Child, Preschool , Female , Genetic Predisposition to Disease , Humans , Infant , Male , Mass Screening , Peptide Fragments , Predictive Value of Tests , Risk Factors , Sensitivity and Specificity
9.
Cell Biochem Biophys ; 48(2-3): 177-82, 2007.
Article in English | MEDLINE | ID: mdl-17709887

ABSTRACT

Even though there are numerous autoantigens for type 1 diabetes, current evidence suggests that a single autoantigen, namely insulin, is responsible for the key initiating event in autoimmunity. If a single autoantigen is necessary for triggering the autoimmune process, then antigen-specific therapy to block or delete the immune response against that autoantigen before epitope spreading occurs, may become a larger focus of future immunotherapeutic strategies. In this article, we review current literature regarding insulin as an autoantigen and potential approaches to deleting insulin-reactive T cells through the use of peptide vaccines and targeted T cell receptor immunizations.


Subject(s)
Autoimmunity/immunology , Diabetes Mellitus, Type 1/immunology , Insulin/immunology , Islets of Langerhans/immunology , Animals , Autoantigens/immunology , Diabetes Mellitus, Type 1/therapy , Epitopes, T-Lymphocyte/immunology , Immunotherapy , Peptide Fragments/immunology , T-Lymphocytes/immunology
10.
J Clin Invest ; 117(7): 1835-43, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17607359

ABSTRACT

NOD mice with knockout of both native insulin genes and a mutated proinsulin transgene, alanine at position B16 in preproinsulin (B16:A-dKO mice), do not develop diabetes. Transplantation of NOD islets, but not bone marrow, expressing native insulin sequences (tyrosine at position B16) into B16:A-dKO mice rapidly restored development of insulin autoantibodies (IAAs) and insulitis, despite the recipients' pancreatic islets lacking native insulin sequences. Splenocytes from B16:A-dKO mice that received native insulin-positive islets induced diabetes when transferred into wild-type NOD/SCID or B16:A-dKO NOD/SCID mice. Splenocytes from mice immunized with native insulin B chain amino acids 9-23 (insulin B:9-23) peptide in CFA induced rapid diabetes upon transfer only in recipients expressing the native insulin B:9-23 sequence in their pancreata. Additionally, CD4(+) T cells from B16:A-dKO mice immunized with native insulin B:9-23 peptide promoted IAAs in NOD/SCID mice. These results indicate that the provision of native insulin B:9-23 sequences is sufficient to prime anti-insulin autoimmunity and that subsequent transfer of diabetes following peptide immunization requires native insulin B:9-23 expression in islets. Our findings demonstrate dependence on B16 alanine versus tyrosine of insulin B:9-23 for both the initial priming and the effector phase of NOD anti-islet autoimmunity.


Subject(s)
Autoimmunity/immunology , Insulin/immunology , Insulin/metabolism , Islets of Langerhans/immunology , Islets of Langerhans/metabolism , Peptide Fragments/immunology , Peptide Fragments/metabolism , Animals , Autoantibodies/immunology , Bone Marrow Transplantation , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Transplantation , Cross-Priming/immunology , Diabetes Mellitus/genetics , Diabetes Mellitus/immunology , Diabetes Mellitus/metabolism , Diabetes Mellitus/pathology , Immunization , Insulin/genetics , Mice , Mice, Inbred NOD , Peptide Fragments/genetics , Spleen/immunology , Spleen/metabolism , Spleen/transplantation , Survival Rate
11.
Diabetes ; 55(7): 1978-84, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16804066

ABSTRACT

A series of recent studies in humans and the NOD mouse model have highlighted the central role that autoimmunity directed against insulin, in particular the insulin B chain 9-23 peptide, may play in the pathogenesis of type 1 diabetes. Both pathogenic and protective T-cell clones recognizing the B:9-23 peptide have been produced. This report describes the successful creation of BDC12-4.1 T-cell receptor (TCR) transgenic mice with spontaneous insulitis in F1 mice (FVB x NOD) and spontaneous diabetes in NOD.RAG(-/-) (backcross 1 generation). Disease progression is heterogeneous and is modified by a series of genetic factors including heterozygosity (H-2(g7)/H-2(q)) versus homozygosity for H-2(g7), the presence of additional T-/B-cell receptor-rearranged genes (RAG(+) versus RAG(-/-)), and the insulin 2 gene knockout (the insulin gene expressed in the NOD thymus). Despite lymphopenia, 40% of H-2(g7/g7) BDC12-4.1 TCR(+) RAG(-/-) Ins2(-/-) mice are diabetic by 10 weeks of age. As few as 13,500 transgenic T-cells from a diabetic TCR(+) RAG(-/-) mouse can transfer diabetes to an NOD.scid mouse. The current study demonstrates that the BDC12-4.1 TCR is sufficient to cause diabetes at NOD backcross 1, bypassing polygenic inhibition of insulitis and diabetogenesis.


Subject(s)
Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus/genetics , Insulin/genetics , Receptors, Antigen, T-Cell/deficiency , Receptors, Antigen, T-Cell/genetics , Adoptive Transfer , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Crosses, Genetic , Diabetes Mellitus/immunology , Female , Genes, RAG-1 , Heterozygote , Interferon-gamma/blood , Male , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, Transgenic
12.
Diabetes ; 54(9): 2549-56, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16123342

ABSTRACT

A number of studies and clinical case reports have implicated interferon (IFN)-alpha as a potential mediator of type 1 diabetes pathogenesis. Administration of polyinosinic:polycytidylic acid (poly I:C), a mimic of viral double-stranded RNA, induces diabetes in C57BL/6 mice expressing the B7.1 costimulatory molecule in islets. We investigated the potential role of IFN-alpha in this disease model. The quantitative correlation between IFN-alpha levels and time to diabetes, diabetes prevention with anti-IFN-alpha antibody, and ability of IFN-alpha itself to induce diabetes are consistent with the hypothesis that poly I:C in this model acts by induction of IFN-alpha in a genetically susceptible host. Numerous recent studies highlight the importance of the innate immune system and toll receptors in determining adaptive immune responses, and we speculate that for type 1 diabetes, viral and other environmental factors may act through induction of IFNs.


Subject(s)
Diabetes Mellitus, Experimental/physiopathology , Interferon-alpha/physiology , Islets of Langerhans/physiopathology , Poly I-C/toxicity , Aging , Animals , CD4-Positive T-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/physiology , Gene Expression/drug effects , Islets of Langerhans/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic
13.
J Pediatr ; 146(4): 494-9, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15812452

ABSTRACT

OBJECTIVES: To assess several transglutaminase autoantibody (TGAA) assays in their ability to distinguish celiac disease (CD) in screening-identified children with abnormal intestine biopsy specimens from those with normal biopsy specimens. STUDY DESIGN: Children at risk for CD (n = 54) composed of type 1 diabetics, first-degree relatives of type 1 diabetics or CD, and HLA-DQ2+ individuals followed from birth received intestine biopsy. Sera obtained at the time of biopsy were tested for TGAA, using the radioimmunoassay and 5 other commercially available enzyme-linked immunosorbent assays. RESULTS: False-positive rates ranged from 28% to 80%. The positive predictive value (PPV) of the tests ranged from 63% to 84% (lower than reported for symptomatic children). Setting a higher cutoff for each assay maximized PPV. CONCLUSIONS: There are significant quantitative differences among all TGAA assays that could affect interpretation of a positive test for CD. The overall false-positive rate for all assays was high in this population. Using the assay as a quantitative rather than qualitative tool by increasing the cutoff of positivity to indicate biopsy increases PPV. Multicenter workshops are needed to identify critical differences and to standardize TGAA assays among laboratories.


Subject(s)
Autoantibodies/blood , Celiac Disease/blood , Celiac Disease/diagnosis , Transglutaminases/immunology , Adolescent , Enzyme-Linked Immunosorbent Assay , Humans , Radioimmunoassay , Sensitivity and Specificity
14.
Ann N Y Acad Sci ; 1037: 193-8, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15699516

ABSTRACT

We hypothesize that NOD mice without native insulin, but with an altered insulin B:9-23 sequence, will be completely protected from diabetes/insulitis if insulin B:9-23 is an essential T cell epitope. To investigate this hypothesis, we have established initial insulin 1- and 2-negative NOD mice with a transgene directing production of preproinsulin with alanine at position B:16 rather than the native tyrosine of both insulin 1 and insulin 2. Sets of primers for PCR-based assays have been created and validated. They are able to distinguish the presence or absence of the insulin gene knockouts and of both native insulin 1 and insulin 2 (and thus distinguish heterozygous versus homozygous knockouts), as well as the presence of the altered insulin transgene, B:16 alanine preproinsulin. Four B:16 alanine transgenic founders were produced directly in NOD mice and, by intercrossing, initial live native insulin-negative B:16 alanine transgenic mice have been generated.


Subject(s)
Alanine/metabolism , Genotype , Insulin/genetics , Proinsulin/genetics , Protein Precursors/genetics , Transgenes , Amino Acid Substitution , Animals , Crosses, Genetic , Female , Heterozygote , Homozygote , Male , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, Transgenic , Microinjections , Ovum/physiology , Promoter Regions, Genetic
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